In the skin model the clonal benign tumor defines the phenotype of the initiated cell. High proliferation, resistance to terminal differentiation and altered gene expression are hallmarks of that phenotype. Our previous studies have revealed that Ras activation is sufficient to produce this complete phenotype and fully initiate tumor formation. Since oncogenic ras is the most frequently detected oncogene in human cancers and frequently associated with early events in epithelial cancer development, the skin model is particularly suited to study the mechanism of its transforming activity. In skin keratinocytes, oncogenic Ras establishes a positive feedback loop through the epidermal growth factor receptor (EGFR), increasing proliferation and simultaneously activating src kinases to tyrosine phosphorylate and inactivate PKC delta, a critical mediator of keratinocyte terminal differentiation. Ras mediated activation of PKCalpha through increased diacylglycerol production contributes to altered expression of keratinocyte differentiation markers by modifying AP-1 activity. By targeting PKCalpha to the epidermis of genetically altered mice, we have shown that as a consequence of Ras action on EGFR and PKCalpha, NFkB translocates to the nucleus of keratinocytes and upregulates the expression and release of CXCR2 ligands. While producing a strong inflammatory response, these ligands also stimulate keratinocyte migration through an autocrine mechanism. Genetic ablation of CXCR2 on ras transduced mouse keratinocytes inhibits migration and precludes tumor formation. The downstream signals from CXCR2 activation in keratinocytes have focused on the activation of AKT, ERK and Src as transducers of the migration signal. In collaboration with medicinal chemists we are developing and testing topical inhibitors of CXCR2 and Src as anti-tumor and anti-inflammation agents. The connection between ras mediated EGFR activation and NFkB translocation has been clarified in collaboration with members of the Trinchieri Laboratory and the CCR Cancer and Inflammation Program. MyD88 is a crucial intermediate in the signaling from pro-inflammatory receptors. Mice ablated for MyD88 or the IL-1 receptor (IL-1R) are resistant to topical skin carcinogenesis, and their ras transduced keratinocytes form only small tumors in orthotopic grafts. In keratinocytes lacking MyD88 or IL-1R, transduction of ras activates the EGFR loop, but these keratinocytes do not upregulate pro-inflammatory ligands. It is now clear that downstream from ras activation of the EGFR is the release of IL-1alpha, and this is the critical element leading to NFkB translocation and induction of an inflammatory cascade essential for tumor formation. These models are being utilized to dissect the contribution of inflammation and autocrine chemokine/cytokine signaling in benign tumor development and to test the effectiveness of IL-1R inhibitors to prevent or reverse ras mediated tumor development. A second model has been developed to study cutaneous inflammation and tumor development. Previously, we described the pro-inflammatory chemotactic action of two highly homologous human S100 proteins (A7 and A15) originally cloned from inflamed psoriatic lesions where they are highly expressed and genetically linked to psoriasis development. We developed discriminating antibodies that reveal their differential expression pattern in psoriasis indicative of their functional diversity. We discovered that RAGE was the receptor for hS100A7 and a G-coupled protein mediated the chemotactic activity of hS100A15. Each protein is proinflammatory and they potentiate inflammation in-vivo. We then cloned the single mouse gene progenitor (mS100A7A15), generated an antibody to the protein and demonstrated mS100A7A15 conserves human properties. As the human counterparts, the mouse progenitor is regulated with epidermal differentiation and highly expressed in well differentiated epithelial tumors. Similarly, the human S100A7 and S100A15 proteins are both upregulated in epithelial breast cancers. Functionally conserved, mS100A7A15 also binds to RAGE and is a chemoattractant. An inducible mS100A7A15 transgene targeted to the mouse epidermis stimulates infiltration of immunocytes and upregulates inflammatory cytokines and chemokines to potentiate inflammation in response to external stimuli. These mice may serve as a model for psoriasis but also will serve as a preclinical model to examine putative inhibitors of RAGE as potential anti-inflammatory agents to prevent tumor promotion. Tumor progression in mouse and human squamous tissues is associated with amplification of EGFR, and this has become a target for therapy. The major dose limiting adverse consequence of anti-EGFR therapy for cancer treatment is a severe cutaneous folliculitis. To model this adverse condition, we have genetically deleted the EGFR in mice or conditionally in mouse skin. In the absence of EGFR, skin tumors differentiate prematurely and do not grow. Microarray analysis of RNA from ras transduced wildtype, EGFR null or EGFR inhibitor treated keratinocytes reveals a 25 gene signature for EGFR ablation in tumor cells. Activation of p38 is a node in this signature, and phospho-p38 is elevated in EGFR null tumors. In this setting of p38 activation, IP10, a chemokine linked to CXCR3 and migration of T cells, dendritic cells and macrophages, is increased. Three isoforms of p38 are expressed in keratinocytes, and current knockdown studies are directed to determine which isoform is activated in response to the inhibition of EGFR. The relevance of these findings to human pathology is emphasized by the phenotype of mice with skin targeted EGFR ablation. Extensive folliculitis and alopecia characterize the dose-limiting skin lesions of patients on EGFR inhibitor drugs and are common finding in adult mice with epidermal ablation of EGFR. Tissue samples isolated from non lesional skin of adult transgenic mice display increased mRNA levels of a subset of inflammatory mediators namely MIP-2, MIP-3alpha, MCP-1, TNFalpha, IL-6, IL-17, IL-18, TARC, TGFbeta, iNOS and G-CSF. In contrast, cutaneous GM-CSF is decreased in mouse and human epidermis where EGFR is suppressed. Targeted knockout mice also show higher plasma levels of IL-17, IL-6, IP-10, MCP-1, TGFbeta and G-CSF. Higher basal and stimulated infiltration of CD45 + cells is detected in EGFR null mouse skin, and mast cells and macrophages are more abundant. Adult double transgenic mice show a higher level of T cell activation (CD69 positive cells) and an increased percentage of gamma-delta T cells and CD11b/Gr1 positive cells harvested from the lymph nodes and the spleen. Treatment of the mice for 3 weeks with a TNFalpha neutralizing drug, Embrel, did not affect the abundance of these cells while treatment with neutralizing G-CSF antibody normalized the percentage of CD11b/Gr1 positive cells and gamma-delta T cells detected in the spleen and lymph nodes. Tamoxifen-regulated conditional skin targeted EGFR null mice are now under study to address the role of modulating EGFR expression during each stage of tumor induction.